PathHunter® Protein Degradation Assay Platform

PathHunter technology incorporates an adaptation of Enzyme Fragment Complementation (EFC) in a novel, cell-based assay format to detect protein degradation. EFC is a homogeneous, non-radioactive detection technology, based on the use of two genetically-engineered β-galactosidase (β-gal) fragments: a large protein fragment (Enzyme Acceptor, EA) and a small peptide fragment (Enzyme Donor, ED). Separately, the β-gal fragments are inactive. When in close proximity in a cell, the enzyme fragments combine to form active β-gal enzyme that hydrolyzes substrate. By using the ProLink/Prolabel detection kit, a chemiluminescent signal is easily detectable.

Technology Principle

HEK cells are engineered to express IκBα with the ProLabel tag attached to the C-terminus of the IκBα. In response to TNFα, endogenous TNFα receptor present in HEK cells signal downstream of IκBα. IκBα protein is degraded in response to NFκB pathway activation. The PathHunter® HEK 293 IκB Functional Assay is used with the PL Detection Kit for measuring human IκB degradation in response to pathway stimulation.

Analysis of TNFα Induced IκB Degradation

PathHunter® HEK IκB cells were plated and stimulated with TNF-α. IκB degradation was measured using the PathHunter® Detection Kit (93-0001) according to the recommended protocol [click graph to enlarge].